Stabilized fibronectin based scaffold molecules

ABSTRACT

Provided herein are proteins comprising a fibronectin based scaffold (FBS) domain, e.g.,  10 Fn3 molecules, that bind specifically to a target, and wherein the FBS domain is linked at its C-terminus to a region consisting of PmXn, wherein P is proline, X is any amino acid and wherein n is 0 or an integer that is at least 1 and m is an integer that is at least 1, and wherein the PmXn moiety provides an enhanced property to the FBS domain, e.g., enhanced stability, relative to the protein that is not linked to the PmXn moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.16/560,521 (now Allowed), filed Sep. 4, 2019, which is a continuation ofU.S. Pat. Application No. 15/127,183 (now U.S. Pat. No.10,450,363),filed Sep. 19, 2016, which is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/US2015/021466, filed Mar. 19, 2015,which claims the benefit of U.S. Provisional Application No. 61/955,975,filed Mar. 20, 2014, and U.S. Provisional Application No. 62/084,270,filed Nov. 25, 2014. The contents of the aforementioned applications arehereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in xml format and is hereby incorporated byreference in its entirety. Said xml copy, created on Jan. 25, 2023 isnamed MXI_553USCN2_Sequence_ Listing.xml and is 190,043 bytes in size.

BACKGROUND

Adnectins are a class of therapeutic proteins with high-affinity andspecific target-binding properties that are derived from the tenth humanfibronectin type III domain (¹⁰Fn3). Whereas wild-type ¹⁰Fn3 isextremely stable and soluble, target-binding variants of ¹⁰Fn3, whichcontain in the order of 4-31 mutations from the wild-type sequence, varywidely in stability and solubility. In other words, any mutations fromthe wild-type ¹⁰Fn3 sequence, even if required for target binding,carries a risk of reducing the stability of protein. As a consequence,it would be desirable to identify modifications that can be made to thewild-type ¹⁰Fn3 sequence that would stabilize it, preferably regardlessof the identity of the residues that mediate Adnectin binding to theirtherapeutic targets.

SUMMARY

Provided herein are stabilized fibronectin based scaffold (FBS)proteins, e.g., Fn3, such as ¹⁰Fn3 molecules (e.g., human ¹⁰Fn3molecules) that are linked at their C-terminus to a moiety consisting ofthe amino acid sequence PmXn, wherein P is proline, X is any amino acid,m is an integer that is at least 1 and n is 0 or an integer that is atleast 1, and wherein the PmXn moiety enhances at least onecharacteristic, e.g., thermostability, of the FBS proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Representation of a crystal structure of the human ¹⁰Fn3 domain(PDB ID: 1FNA), and the protein sequence of the polypeptide visible inthe structure. The last two residues defined in the structure, the “EI”in the sequence of interest, are shown as black spheres, immediatelydownstream from the C-terminal beta strand, G.

DETAILED DESCRIPTION Definitions

An “amino acid residue” is the remaining portion of an amino acid aftera water molecule has been lost (an H+ from the nitrogenous side and anOH— from the carboxylic side) in the formation of a peptide bond.

As used herein, a “¹⁰Fn3 domain” or “¹⁰Fn3 moiety” or “¹⁰Fn3 molecule”refers to wild-type ¹⁰Fn3 and biologically active variants thereof,e.g., biologically active variants that specifically bind to a target,such as a target protein. A wild-type human ¹⁰Fn3 domain may compriseone of the amino acid sequences set forth in SEQ ID NO: 1-8.Biologically active variants of a wild-type human ¹⁰Fn3 domain include¹⁰Fn3 domains that comprise at least, at most or about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40 or 45 amino acid changes, i.e., substitutions, additions ordeletions, relative to a ¹⁰Fn3 domain comprising any one of SEQ ID NOs:1-8. A biologically active variant of a wild-type ¹⁰Fn3 domain may alsocomprise, or comprise at most, 1-3, 1-5, 1-10, 1-15, 1-10, 1-25, 1-30,1-35, 1-40 or 1-45 amino acid changes relative to a ¹⁰Fn3 domaincomprising any one of SEQ ID NOs: 1-8. In certain embodiments, abiologically active variant of a wild-type ¹⁰Fn3 domain does notcomprise more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or 45 amino acidchanges, i.e., substitutions, additions or deletions, relative to an¹⁰Fn3 domain comprising any one of SEQ ID NOs: 1-8. Amino acid changesmay be in a loop region, in a strand or in the N-terminal or C-terminalregion. Exemplary degenerate ¹⁰Fn3 amino acid sequences allowing foramino acid changes in the loop regions are provided herein as SEQ IDNOs: 9-16.

By “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

A “region” of a ¹⁰Fn3 domain (or moiety or molecule) as used hereinrefers to either a loop (AB, BC, CD, DE, EF and FG), a β-strand (A, B,C, D, E, F and G), the N-terminus (corresponding to amino acid residues1-7 of SEQ ID NO: 1), or the C-terminus (corresponding to amino acidresidues 93-94 of SEQ ID NO: 1).

A “north pole loop” of a ¹⁰Fn3 domain (or moiety) refers to any one ofthe BC, DE and FG loops of a ¹⁰Fn3 domain.

A “south pole loop” of a ¹⁰Fn3 domain (or moiety) refers to any one ofthe AB, CD and EF loops of a ¹⁰Fn3 domain.

A “scaffold region” refers to any non-loop region of a human ¹⁰Fn3domain. The scaffold region includes the A, B, C, D, E, F and Gβ-strands as well as the N-terminal region (amino acids corresponding toresidues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acidscorresponding to residues 93-94 of SEQ ID NO: 1).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST℠,BLAST℠-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by a sequence alignment program, such asBLAST℠, BLAST℠-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®), in thatprogram’s alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A.

As used herein, an amino acid residue in a polypeptide is considered to“contribute to binding” a target if (1) any of the non-hydrogen atoms ofthe residue’s side chain or main chain is found to be within fiveangstroms of any atom of the binding target based on an experimentallydetermined three-dimensional structure of the complex, and/or (2)mutation of the residue to its equivalent in wild-type ¹⁰Fn3 (e.g., SEQID NO: 1), to alanine, or to a residue having a similarly sized orsmaller side chain than the residue in question, leads to a measuredincrease of the equilibrium dissociation constant to the target (e.g.,an increase in the k_(on)).

The serum or plasma “half-life” of a polypeptide can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thepolypeptide and/or clearance or sequestration of the polypeptide bynatural mechanisms. The half-life can be determined in any manner knownper se, such as by pharmacokinetic analysis. Suitable techniques will beclear to the person skilled in the art, and may, for example, generallyinvolve the steps of administering a suitable dose of a polypeptide to aprimate; collecting blood samples or other samples from said primate atregular intervals; determining the level or concentration of thepolypeptide in said blood sample; and calculating, from (a plot of) thedata thus obtained, the time until the level or concentration of thepolypeptide has been reduced by 50% compared to the initial level upondosing. Methods for determining half-life may be found, for example, inKenneth et al., Chemical Stability of Pharmaceuticals: A Handbook forPharmacists (1986); Peters et al., Pharmacokinete Analysis: A PracticalApproach (1996); and Gibaldi, M. et al., Pharmacokinetics, Second Rev.Edition, Marcel Dekker (1982).

Serum half-life can be expressed using parameters such as the t½-alpha,t½-beta and the area under the curve (AUC). An “increase in half-life”refers to an increase in any one of these parameters, any two of theseparameters, or in all three these parameters. In certain embodiments, anincrease in half-life refers to an increase in the t½-beta, either withor without an increase in the t½-alpha and/or the AUC or both.

“Shelf-life” of a pharmaceutical product, e.g., a protein comprising anFBS moiety and an HSA moiety, is the length of time the product isstored before decomposition occurs. For example, shelf-life may bedefined as the time for decomposition of 0.1%, 0.5%, 1%, 5%, or 10% ofthe product.

Overview

Provided herein are proteins comprising a fibronectin based scaffold(FBS) domain, e.g., Fn3, such as ¹⁰Fn3 molecules, that bind specificallyto a target, and wherein the FBS domain is linked at its C-terminus to aregion consisting of PmXn, wherein P is proline, X is any amino acid andwherein n is 0 or an integer that is at least 1 and m is an integer thatis at least 1. The application is based at least in part on thediscovery that adding a proline and optionally one or more amino acidsat the C-terminus of a ¹⁰Fn3 molecule increases at least onecharacteristic of the ¹⁰Fn3 molecule, e.g., its thermostability orsolubility, relative to the unmodified ¹⁰Fn3 molecule.

The ¹⁰Fn3 molecules described herein may be designed to bind to anytarget of interest. In exemplary embodiments, the target is an antigen,a polypeptide or a therapeutic protein target of interest. Exemplarytherapeutically desirable targets, include, for example, tumor necrosisfactor alpha (TNF-alpha), VEGFR2, PCSK9, IL-23, EGFR and IGF1R.

Fibronectin Based Scaffolds

As used herein, a “fibronectin based scaffold” or “FBS” protein ormoiety refers to proteins or moieties that are based on a fibronectintype III (“Fn3”) repeat. Fn3 is a small (about 10 kDa) domain that hasthe structure of an immunoglobulin (Ig) fold (i.e., an Ig-likeβ-sandwich structure, consisting of seven β-strands and six loops).Fibronectin has 18 Fn3 repeats, and while the sequence homology betweenthe repeats is low, they all share a high similarity in tertiarystructure. Fn3 domains are also present in many proteins other thanfibronectin, such as adhesion molecules, cell surface molecules, e.g.,cytokine receptors, and carbohydrate binding domains. For reviews seeBork et al., Proc. Natl. Acad. Sci. USA, 89(19):8990-8994 (1992); Borket al., J. Mol. Biol., 242(4):309-320 (1994); Campbell et al.,Structure, 2(5):333-337 (1994); Harpez et al., J. Mol. Biol.,238(4):528-539 (1994)). The term “FBS” protein or moiety is intended toinclude scaffolds based on Fn3 domains from these other proteins (i.e.,non fibronectin molecules).

An Fn3 domain is small, monomeric, soluble, and stable. It lacksdisulfide bonds and, therefore, is stable under reducing conditions. Fn3domains comprise, in order from N-terminus to C-terminus, a beta orbeta-like strand, A; a loop, AB; a beta or beta-like strand, B; a loop,BC; a beta or beta-like strand, C; a loop, CD; a beta or beta-likestrand, D; a loop, DE; a beta or beta-like strand, E; a loop, EF; a betaor beta-like strand, F; a loop, FG; and a beta or beta-like strand, G.The seven antiparallel β-strands are arranged as two beta sheets thatform a stable core, while creating two “faces” composed of the loopsthat connect the beta or beta-like strands. Loops AB, CD, and EF arelocated at one face (“the south pole”) and loops BC, DE, and FG arelocated on the opposing face (“the north pole”).

The loops in Fn3 molecules are structurally similar to complementarydetermining regions (CDRs) of antibodies, and when altered, may beinvolved in binding of the Fn3 molecule to a target, e.g., a targetprotein. Other regions of Fn3 molecules, such as the beta or beta-likestrands and N-terminal or C-terminal regions, when altered, may also beinvolved in binding to a target. Any or all of loops AB, BC, CD, DE, EFand FG may participate in binding to a target. Any of the beta orbeta-like strands may be involved in binding to a target. Fn3 domainsmay also bind to a target through one or more loops and one or more betaor beta-like strands. Binding may also require the N-terminal orC-terminal regions. An FBS domain for use in a protein may comprise allloops, all beta or beta-like strands, or only a portion of them, whereincertain loops and/or beta or beta-like strands and/or N- or C-terminalregions are modified (or altered), provided that the FBS domainpreferably binds specifically to a target. For example, an FBS domainmay comprise 1, 2, 3, 4, 5 or 6 loops, 1, 2, 3, 4, 5, 6, 7, or 8 betastrands, and optionally an N-terminal and/or C-terminal region, whereinone or more loops, one or more beta strands, the N-terminal regionand/or the C-terminal regions are modified relative to the wild-type FBSdomain.

In exemplary embodiments, ligand (or target) binding FBS moietiesdescribed herein are based on the tenth fibronectin type III domain,i.e., the tenth module of Fn3 (¹⁰Fn3). The amino acid sequence of awild-type human ¹⁰Fn3 moiety is as follows:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1) (the AB, CD andEF loops are underlined; the BC, FG, and DE loops are emphasized inbold; the β-strands are located between or adjacent to each of the loopregions; and the N-terminal region is shown in italics). The last twoamino acid residues of SEQ ID NO: 1 are a portion of a C-terminalregion.

Wild-type human ¹⁰Fn3 molecules also include those lacking theN-terminal region or a portion thereof. For example, a wild-type ¹⁰Fn3molecule may comprise SEQ ID NO: 1, wherein amino acid residues 1, 1-2,1-3, 1-4, 1-5, 1-6 or 1-7 are deleted (SEQ ID NOs: 2-8, respectively).Table 1 shows the amino acid sequence of these wild-type human ¹⁰Fn3moieties:

TABLE 1 Amino acid sequences of wild-type human ¹⁰Fn3 molecules withvarious N-terminal regions Version N-terminal region Wild-type human¹⁰Fn3 core domain Full length 1 VSDVPRD (SEQ ID NO : 19)LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 1 2 SDVPRD (SEQ ID NO : 20)LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQID NO: 17) SEQ ID NO: 2 3 DVPRD (SEQ ID NO : 21)LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 3 4 VPRD (SEQ ID NO : 22)LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 4 5 PRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 5 6 RDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 6 7 DLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 7 8 -LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(SEQ ID NO: 17) SEQ ID NO: 8

In some embodiments, the AB loop corresponds to residues 14-17, the BCloop corresponds to residues 23-31, the CD loop corresponds to residues37-47, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 63-67, and the FG loop corresponds to residues75-87 of SEQ ID NO: 1. The BC, DE and FG loops align along one face ofthe molecule, i.e., the “north pole”, and the AB, CD and EF loops alignalong the opposite face of the molecule, i.e., the “south pole”. In SEQID NO: 1, β-strand A corresponds to residues 8-13, β-strand Bcorresponds to residues 18-22, β-strand C corresponds to residues 32-36,beta strand D corresponds to residues 48-50, β-strand E corresponds toresidues 57-62, β-strand F corresponds to residues 68-74, and β-strand Gcorresponds to residues 88-92. The β-strands are connected to each otherthrough the corresponding loop, e.g., strands A and B are connected vialoop AB in the formation β-strand A, loop AB, β-strand B, etc.

An example of FBS proteins that are based on human ¹⁰Fn3 domains areadnectins (Adnexus, a wholly owned subsidiary of Bristol-Myers Squibb).Adnectins are ¹⁰Fn3 molecules in which CDR-like loop regions, β-strands,N-terminal and/or C-terminal regions of a ¹⁰Fn3 domain has been modifiedto evolve a protein capable of binding to a compound of interest. Forexample, U.S. Pat. No. 7,115,396 describes ¹⁰Fn3 domain proteins whereinalterations to the BC, DE, and FG loops result in high affinity TNFαbinders. U.S. Pat. No. 7,858,739 describes Fn3 domain proteins whereinalterations to the BC, DE, and FG loops result in high affinity VEGFR2binders.

In certain embodiments, the FBS moiety comprises a ¹⁰Fn3 domain that isdefined generally by the following degenerate sequence:

VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 9),

or by a sequence selected from the group of SEQ ID NO: 10-16, whichsequences are identical to SEQ ID NO: 9, except that they are lacking 1,2, 3, 4, 5, 6 or 7 N-terminal amino acids, respectively. Table 2 showsthe amino acid sequences of these degenerate human ¹⁰Fn3 molecules.

TABLE 2 Amino acid sequences of degenerate wild-type human ¹⁰Fn3molecules with various N-terminal regions Version N-terminal regionDegenerate wild-type human ¹⁰Fn3 core domain Full length 1 VSDVPRD (SEQID NO : 19) LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 9 2SDVPRD (SEQ ID NO : 20) LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 103 DVPRD (SEQ ID NO : 21) LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 114 VPRD (SEQ ID NO : 22) LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 125 PRD LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 136 RD LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 147 D LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 158 - LEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 18) SEQ ID NO: 16

In SEQ ID NOs: 25-32 and 50, the AB loop is represented by (X)_(u), theBC loop is represented by (X)_(v), the CD loop is represented by(X)_(w), the DE loop is represented by (X)_(x), the EF loop isrepresented by (X)_(y) and the FG loop is represented by X_(z). Xrepresents any amino acid and the subscript following the X representsan integer of the number of amino acids. In particular, u, v, w, x, yand z may each independently be anywhere from 2-20, 2-15, 2-10, 2-8,5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 aminoacids. The sequences of the beta strands (underlined in SEQ ID NO: 9)may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5,from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,deletions or additions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NOs: 9-16. In someembodiments, the sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 substitutions, e.g., conservativesubstitutions, across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 9-16.

In certain embodiments, the hydrophobic core amino acid residues (boldedresidues in SEQ ID NO: 9 above) are fixed, and any substitutions,conservative substitutions, deletions or additions occur at residuesother than the hydrophobic core amino acid residues. Thus, in someembodiments, the hydrophobic core residues of the polypeptides providedherein have not been modified relative to the wild-type human ¹⁰Fn3domain (e.g., SEQ ID NO: 1).

In some embodiments, an FBS moiety comprises a ¹⁰Fn3 domain, wherein the¹⁰Fn3 domain comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; aloop, EF; and a loop, FG; and has at least one loop selected from loopAB, BC, CD, DE, EF and FG with an altered amino acid sequence relativeto the sequence of the corresponding loop of the wild-type human ¹⁰Fn3domain. In some embodiments, a single loop is altered. In someembodiments, at most 2 loops are altered. In some embodiments, at most 3loops are altered. In some embodiments, the BC, DE and/or FG loops arealtered. In certain embodiments, the AB, CD and EF loops are altered. Incertain embodiments, the FG loop is the only loop that is altered. Incertain embodiments, the CD loop is the only loop that is altered. Inother embodiments, the CD and FG loops are both altered, and optionally,no other loops are altered. In certain embodiments, the CD and EF loopsare both altered, and optionally, no other loops are altered. In someembodiments, one or more specific scaffold alterations are combined withone or more loop alterations. By “altered” is meant one or more aminoacid sequence alterations relative to a template sequence (i.e., thecorresponding wild-type human fibronectin domain) and includes aminoacid additions, deletions, and substitutions. Exemplary ¹⁰Fn3 moleculescomprising specific combinations of altered loops and/or scaffoldregions (e.g., beta strands, N-terminal region and C-terminal region)are further disclosed herein.

It should be understood that not every residue within a loop regionneeds to be modified in order to achieve a ¹⁰Fn3 binding domain havingstrong affinity for a desired target. Additionally, insertions anddeletions in the loop regions may also be made while still producinghigh affinity ¹⁰Fn3 binding domains.

In some embodiments, one or more loops selected from AB, BC, CD, DE, EFand FG may be extended or shortened in length relative to thecorresponding loop in wild-type human ¹⁰Fn3. In any given polypeptide,one or more loops may be extended in length, one or more loops may bereduced in length, or combinations thereof. In some embodiments, thelength of a given loop may be extended by 2-25, 2-20, 2-15, 2-10, 2-5,5-25, 5-20, 5-15, 5-10, 10-25, 10-20, or 10-15 amino acids. In someembodiments, the length of a given loop may be reduced by 1-15, 1-11,1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. In particular, the FGloop of ¹⁰Fn3 is 13 residues long, whereas the corresponding loop inantibody heavy chains ranges from 4-28 residues. To optimize antigenbinding in polypeptides relying on the FG for target binding, therefore,the length of the FG loop of ¹⁰Fn3 may be altered in length as well asin sequence to obtain the greatest possible flexibility and affinity intarget binding.

In some embodiments, the FBS moiety comprises a ¹⁰Fn3 domain wherein thenon loop regions comprise an amino acid sequence that is at least 80,85, 90, 95, 98, or 100% identical to the non-loop regions of SEQ ID NO:1, wherein at least one loop selected from AB, BC, CD, DE, EF and FG isaltered. For example, in certain embodiments, the AB loop may have up to4 amino acid substitutions, up to 10 amino acid insertions, up to 3amino acid deletions, or a combination thereof; the BC loop may have upto 10 amino acid substitutions, up to 4 amino acid deletions, up to 10amino acid insertions, or a combination thereof; the CD loop may have upto 6 amino acid substitutions, up to 10 amino acid insertions, up to 4amino acid deletions, or a combination thereof; the DE loop may have upto 6 amino acid substitutions, up to 4 amino acid deletions, up to 13amino acid insertions, or a combination thereof; the EF loop may have upto 5 amino acid substitutions, up to 10 amino acid insertions, up to 3amino acid deletions, or a combination thereof; and/or the FG loop mayhave up to 12 amino acid substitutions, up to 11 amino acid deletions,up to 25 amino acid insertions, or a combination thereof.

In some embodiments, an FBS moiety comprises a ¹⁰Fn3 domain having atleast 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity to ahuman ¹⁰Fn3 domain having an amino acid sequence selected from the groupof sequence comprising SEQ ID NOs: 1-16. In certain embodiments, the FBSmoiety provided herein has at least 50% identity to an amino acidsequence selected from the group of amino acid sequences comprising SEQID NO: 1-16. In other embodiments, the FBS moiety has at least 65%identity to an amino acid sequence selected from the group of amino acidsequences comprising SEQ ID NO: 1-16. In certain embodiments, one ormore of the loops will not be modified relative to the sequence of thecorresponding loop of the wild-type sequence and/or one or more of theβ-strands will not be modified relative to the sequence of thecorresponding β-strand of the wild-type sequence and/or the N-terminalor C-terminal regions will not be modified. In certain embodiments, eachof the beta or beta-like strands of a ¹⁰Fn3 domain in an FBS moiety maycomprise, consist essentially of, or consist of an amino acid sequencethat is at least 80%, 85%, 90%, 95% or 100% identical to the sequence ofa corresponding beta or beta-like strand of SEQ ID NO: 1. Preferably,variations in the β-strand regions will not disrupt the stability of thepolypeptide in physiological conditions.

In some embodiments, the non-loop region of a ¹⁰Fn3 domain may bemodified by one or more conservative substitutions. As many as 5%, 10%,20% or even 30% or more of the amino acids in the ¹⁰Fn3, domain may bealtered by a conservative substitution without substantially alteringthe affinity of the ¹⁰Fn3 for a ligand. In certain embodiments, thenon-loop regions, e.g., the β-strands may comprise anywhere from 0-15,0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3,2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15, or 5-10 conservative amino acidsubstitutions. In exemplary embodiments, the scaffold modification mayreduce the binding affinity of the ¹⁰Fn3 binder for a ligand by lessthan 100-fold, 50-fold, 25-fold, 10-fold, 5-fold, or 2-fold. It may bethat such changes may alter the immunogenicity of the ¹⁰Fn3 in vivo, andwhere the immunogenicity is decreased, such changes may be desirable. Asused herein, “conservative substitutions” are residues that arephysically or functionally similar to the corresponding referenceresidues. That is, a conservative substitution and its reference residuehave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like. Exemplaryconservative substitutions include those fulfilling the criteria definedfor an accepted point mutation in Dayhoff et al., Atlas of ProteinSequence and Structure, 5:345-352 (1978 and Supp.). Examples ofconservative substitutions include substitutions within the followinggroups: (a) valine, glycine; (b) glycine, alanine; (c) valine,isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine,glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and(h) phenylalanine, tyrosine.

Also provided herein are ¹⁰Fn3 domains having combinations of loop andscaffold modifications. Conjugates may comprise a ¹⁰Fn3, domaincomprising (i) a modification in the amino acid sequence of at least oneof loops AB, BC, CD, DE, EF, or FG, and (ii) a modification in the aminoacid sequence of at least one scaffold region (i.e., a modification inat least one β-strand, the N-terminal region, and/or the C-terminalregion), wherein the modified loop(s) and modified scaffold region(s)both contribute to binding the same target. In exemplary embodiments,the scaffold region modifications are located adjacent to modificationsin a loop region, e.g., if the AB loop is modified, scaffold mutationsmay tend to be located in β-strand A and/or β-strand B, which areadjacent to the AB loop in the linear sequence of the ¹⁰Fn3 domain. Inother embodiments, a cluster of modifications may be found together inloop and scaffold regions that are adjacent to one another in the linearsequence of the Fn3 domain. For example, Fn3 binders having both loopand scaffold modifications, may have clusters of amino acidmodifications in the following combinations of loop and scaffold regionsthat are adjacent to each other in the linear sequence of the Fn3domain: β-strand/loop/ β-strand, loop/ β-strand/loop, loop/β-strand/loop/ β-strand, terminal region/ β-strand/loop, or loop/β-strand/terminal region, etc. For example, Fn3 domains having novelcombinations of loop and scaffold modifications may have clusters ofmodifications such that over a stretch of 20 contiguous amino acids atleast 15 of the amino acids are modified relative to wild-type. In otherembodiments, at least 17 out of 20, 18 out of 20, 17 out of 25, 20 outof 25, or 25 out of 30 residues in a contiguous stretch are modifiedrelative to the wild-type Fn3 domain sequence over the correspondingstretch of amino acids. In certain embodiments, a given Fn3 domain mayhave two or three clusters of modifications separated by stretches ofunmodified (i.e., wild-type) sequence. For any given region (i.e., aloop, β-strand or terminal region) that is modified, all or only aportion of the region may be modified relative to the wild-typesequence. When a β-strand region is modified, preferably the hydrophobiccore residues remain unmodified (i.e., wild-type) and one or more of thenon-core residues in the β-strand are modified.

In some embodiments, ¹⁰Fn3 domains comprise a binding face along the“west-side” of the molecule (“West-side binders” or “WS binders”). WSbinders may comprise a modified CD loop and a modified FG loop, ascompared to the corresponding CD and FG loop sequences set forth in SEQID NO: 1. The CD loop and the FG loop both contribute to binding to thesame target. In certain embodiments, the WS binders may compriseadditional modifications at one or more regions within the Fn3 domain.For example, WS binders may comprise scaffold modifications in one ormore of the β-strand regions adjacent to the CD and/or FG loops. Inparticular, WS binders may comprise sequence modifications in one ormore of β-strand C, β-strand D, β-strand F, and/or β-strand G. Exemplaryscaffold modifications include modifications at one or more scaffoldregion positions corresponding to the amino acid positions: 33, 35, 49,69, 71, 73, 89 and/or 91 of SEQ ID NO: 1. The WS binders may alsocomprise modifications in the BC loop, particularly in the C-terminalportion of the BC loop. In one embodiment, the last two residues of theBC loop (i.e., corresponding to amino acids 30 and 31 in the wild-type¹⁰Fn3 domain) are modified relative to the wild-type sequence. All or aportion of the additional loop and scaffold modifications may contributeto binding to the target in conjunction with the modified CD and FGloops. Preferably, the hydrophobic core residues are not modifiedrelative to the wild-type sequence.

Exemplary WS binders include those having a wild-type or mutated aminoacid at positions 30, 31, 33, 35, 37, 38, 46, 47, 49, 50, 67, 69, 71,73, 75, 76, 84, 85, 86, 87, 89 or 91.

In some embodiments, a ¹⁰Fn3 domain comprises modifications in the CD,DE and, in some cases, EF loops, wherein the loop modifications allcontribute to target binding. These polypeptides are referred to as“front binders”. The front binders may additionally comprisemodifications in one or more scaffold regions, particularly in scaffoldregions that flank or are adjacent to a modified loop region. Forexample, the front binders may comprise a scaffold modification in oneor more of β-strand C, β-strand D, and/or β-strand E relative to thesequences of the corresponding β-strands of the wild-type Fn3 domain,e.g., human ¹⁰Fn3 domain (SEQ ID NO: 1). Preferably the hydrophobic coreresidues are not modified relative to the wild-type sequence. Exemplaryscaffold modifications that may be present in front binders, includemodifications at one or more positions corresponding to amino acidpositions 36, 49, 58 and/or 50 of SEQ ID NO: 1. Such scaffoldmodifications may contribute to binding to the target together with themodified loops. In certain embodiments, the front binders may compriseclusters of modifications spanning several loop and strand regions ofthe Fn3, e.g., ¹⁰Fn3, domain. In particular, the front binders maycomprise modifications in at least 15, 20, 24, 25, or 27 of the 31residues between the amino acids corresponding to residues 36 through 66of the wild-type Fn3, e.g., human ¹⁰Fn3, domain (SEQ ID NO: 1). The loopand/or strand modifications may include amino acid substitutions,deletions and/or insertions, or combinations thereof. In exemplaryembodiments, the CD loop is extended in length or reduced in lengthrelative to the CD loop of the Fn3, e.g., wild-type human ¹⁰Fn3, domain(SEQ ID NO: 1).

In some embodiments, ¹⁰Fn3 domains comprise modifications in the EF andFG loops, wherein the loop modifications contribute to binding the sametarget. These polypeptides are referred to as “back binders” herein. Theback binders may comprise additional modifications in other loop and/orscaffold regions. For example, a back binder may contain modificationsin at least a portion of the AB loop, preferably the N-terminal portionof the AB loop. In an exemplary embodiment, the first two amino acids ofthe AB loop (i.e., corresponding to amino acid residues 14 and 15 of thewild-type ¹⁰Fn3 domain) are modified relative to the wild-type sequence.In certain embodiments, a back binder may also contain one or morescaffold modifications, particularly modifications in one or morescaffold regions that are adjacent to a modified loop region. Forexample, back binders may contain one or more modifications in one ormore of β-strand A, β-strand G, the N-terminal region, and/or theC-terminal region. Preferably the hydrophobic core residues are notmodified relative to the wild-type sequence. Exemplary scaffoldmodifications include modifications at one or more positionscorresponding to amino acid positions 1-7, 9-13, 89, 91, 93 and/or 94 ofSEQ ID NO: 1. One or more of the additional loop and/or scaffoldmodifications may contribute to binding to the target along with themodified EF and FG loops. Suitable loop and/or scaffold regionmodifications include amino acid substitutions, deletions and/orinsertions, or combinations thereof. In certain embodiments, the aminoacid sequence of the FG loop is extended in length or reduced in lengthrelative to the FG loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO:1).

In certain embodiments, a back binder may comprise a cluster of modifiedamino acid residues over a contiguous span of several regions in the¹⁰Fn3 domain. For example, at least 14 of the first 15 amino acidresidues of the Fn3, e.g., ¹⁰Fn3, domain may be modified relative to thecorresponding residues in the wild-type Fn3, e.g., human ¹⁰Fn3, domain(SEQ ID NO: 1), and/or at least 15 of the 18 residues between the aminoacids corresponding to residues 80 through 97 (or 94) of the wild-typeFn3, e.g., human ¹⁰Fn3, domain (SEQ ID NO: 1 or 23) may be modifiedrelative to the corresponding residues in the wild-type sequence. Whenreferring to amino acids at positions further C-terminal to 94 in a¹⁰Fn3 molecule, it is in the context of a ¹⁰Fn3 molecule that comprisesthe flexible linker between the 10^(th) and 11^(th) repeat of the Fn3domain, i.e., EIDKPSQ (SEQ ID NO: 113), thus forming a 101 amino acidlong protein. Thus, SEQ ID NO: 1 linked to EIDKPSQ (SEQ ID NO: 113) atits C-terminus is represented by SEQ ID NO: 23.

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT EIDKP SQ (SEQ IDNO: 23)

In certain embodiments, a ¹⁰Fn3 domain comprises modifications in theamino acid sequences of β-strand A, loop AB, β-strand B, loop CD,β-strand E, loop EF, and β-strand F, relative to the sequences of thecorresponding regions of the wild-type sequence. These polypeptides arereferred to as “south pole binders” or “SP binders” herein. The modifiedloops and strands contribute to binding to the same target. The aminoacid sequence of the CD loop may be extended in length or reduced inlength relative to the CD loop of the wild-type Fn3, e.g., human ¹⁰Fn3,domain (SEQ ID NO: 1 or 23). The south pole binders may compriseadditional modifications in β-strand G and/or the C-terminal regionrelative to the sequence of the corresponding region of the wild-typesequence. In exemplary embodiments, the south pole binders may compriseone or more modifications at amino acids corresponding to positions 11,12, 19, 60, 61, 69, 91, 93 and 95-97 of the wild-type sequence.

In some embodiments, a ¹⁰Fn3 domain comprises modified BC, DE and FGloops, as compared to the corresponding BC, DE and FG loop sequences setforth in SEQ ID NO: 1 or 23, as well as additional modifications in oneor more of β-strand C, β-strand D, β-strand F and β-strand G strandresidues. The β-strand and loop region modifications together contributeto binding to the target. These proteins are referred to as “Northwestbinders”, or “NW binders”, herein. In exemplary embodiments, the NWbinders comprise one or more scaffold modifications at any one of, orcombination of, amino acid positions corresponding to scaffold regionpositions R33, T49, Y73 and S89 of SEQ ID NO: 1 or 23. Suitablemodifications in loop and scaffold regions include amino acidsubstitutions, deletions and/or insertions, or combinations thereof. Incertain embodiments, one or more of the BC, DE and FG loops are extendedin length or reduced in length, or combinations thereof, relative to thewild-type sequence. In one embodiment, each of the BC, DE and FG loopsare extended in length or reduced in length, or combinations thereof,relative to the wild-type sequence (e.g., SEQ ID NO: 1 or 23). Incertain embodiments, only a portion of the BC loop is modified,particularly the C-terminal portion, relative to the wild-type sequence.For example, the BC loop may be modified only at amino acid residuescorresponding to amino acids 27-31 of the wild-type BC loop, whereas therest of the BC loop (i.e., corresponding to residues 23-26 of thewild-type loop) are left unmodified.

In some embodiments, a ¹⁰Fn3 domain comprises a modified BC, DE and FGloop as well as one or more additional modifications in any one of, orcombination of, the N-terminal region, β-strand A, β-strand B and/orβ-strand E. These proteins are referred to as “Northeast binders”, or“NE binders”, herein. In exemplary embodiments, the NE binders aremodified at any one of, or combination of, amino acids corresponding toscaffold region positions 1-7, E9, L19, S21 and/or T58 of the wild-typesequence (SEQ ID NO: 1 or 23). The combination of modified loop andscaffold regions contributes to binding to the target.

In some embodiments, a ¹⁰Fn3 domain comprises modifications in one ormore of the AB, CD, DE and EF loops, as well as additional modificationsin one or more of β-strand B, β-strand D and/or β-strand E. Theseproteins are referred to as “South Front binders” herein. Thecombination of modified loop and strand residues contributes to bindingto the target. In exemplary embodiments, a South Front binder may bemodified at one or more amino acid positions corresponding to scaffoldregion positions L19, T49, T58, S60, and/or G61 of SEQ ID NO: 1 or 23and/or at one or more amino acid positions corresponding to loop regionpositions T14-S17, P51, T56, G40-E47, and/or K63-G65 of SEQ ID NO: 1 or23. In exemplary embodiments, a South Front binder may be extended inlength or reduced in length in the AB loop, between amino acidscorresponding to residues 18 and 20 of the wild-type sequence, and/or inthe CD loop.

In some embodiments, a ¹⁰Fn3 domain comprises a modified β-strand A andβ-strand G, as compared to the corresponding strand of SEQ ID NO: 1 or23. These proteins are referred to as “AG Binders” or “AG Strand”binders herein. In certain embodiments, the AG strand binders compriseclusters of modifications at the N-terminal and C-terminal portions ofthe Fn3, e.g., ¹⁰Fn3, domain, whereas the middle portion of the Fn3remains unmodified. For example, an AG strand binder may comprisemodifications at 16 out of 19 of the first 19 amino acids in the ¹⁰Fn3domain (i.e., corresponding to amino acid positions 1-19 of SEQ ID NO: 1or 23) and modifications at 13-17 out of 18 of the last 18 amino acidsin the ¹⁰Fn3 domain (i.e., corresponding to amino acid positions 84-101of SEQ ID NO: 9) or at 14-18 out of 22 of the last 22 amino acids in the¹⁰Fn3 domain (i.e., corresponding to amino acid positions 80-101 of SEQID NO: 9). In exemplary embodiments, an AG binder may comprisemodifications at one or more positions corresponding to positions 1-7,9, 11-17, 19, 84-89 and 91-97 of SEQ ID NO: 9. Preferably the modifiedregions in an AG binder contribute to binding to the same target.

In some embodiments, a ¹⁰Fn3 domain comprises a modified CD and EF loop,as well as additional modifications in any one of, or combination ofresidues corresponding to positions 69 or 91-97 of SEQ ID NO: 1 or 23.These proteins are referred to as “Southwest binders”, or “SW binders”,herein. The modified loop and scaffold regions contribute to binding tothe target.

In certain embodiments, proteins comprise a ¹⁰Fn3 domain having reducedimmunogenicity, wherein a portion of the BC loop is left as wild-type.Preferably such polypeptides have lower immunogenicity relative to anequivalent polypeptide with modifications in a greater portion of the BCloop. In exemplary embodiments, the N-terminal portion of the BC loop isleft as wild-type. For example, the first 1, 2, 3, 4, 5, or 5 residuesof the BC loop may be left as wild-type, while the remaining C-terminalresidues of the BC loop can be modified. In Fn3 designs having at leasta portion of the N-terminal region of the BC loop as wild-type, it maybe desirable to leave all or a portion of β-strand B and/or β-strand Cunmodified relative to the wild-type sequence as well, particularly theportions of β-strand B and/or β-strand C that are adjacent to the BCloop (i.e., the C-terminal portion of β-strand B and/or the N-terminalportion of β-strand C). In exemplary embodiments, Fn3 domains having thewild-type sequence in an N-terminal portion of the BC loop and reducedimmunogenicity may not have any modifications in the N-terminal region,β-strand A, AB loop, and β-strand B. In Fn3 designs with a portion ofthe BC loop as wild-type, the modified portion of the BC loop maycontribute to target binding along with modifications in other regionsof the ¹⁰Fn3 domain.

In certain embodiments, proteins comprise a ¹⁰Fn3 domain having reducedimmunogenicity, wherein the strong HLA anchor in the region of β-strandB/BC loop/β-strand C (the “BC anchor”) has been removed or destroyed(e.g., modified relative to the wild-type sequence in a manner thatreduces binding affinity to one or more HLA receptors). For example, theBC anchor may be removed or destroyed by modifying the Fn3, e.g., ¹⁰Fn3,domain at one or more positions corresponding to positions L19, S21, R33and/or T35 of SEQ ID NO:1. When the BC anchor has been removed ordestroyed, it is possible to modify the sequence of the BC loop withoutsignificantly increasing the immunogenic potential of the BC region.Accordingly, many such Fn3 designs have modifications in the BC loop inaddition to the modifications in β-strand B and/or β strand C. The BCloop may contribute to target binding, optionally in combination withmodifications in other regions of the Fn3 domain. The modifications inβ-strand B and/or β-strand C may or may not contribute to targetbinding.

In exemplary embodiments, an FBS, e.g., a ¹⁰Fn3 domain, binds to adesired target with a K_(d) of less than 500 nM, 100 nM, 50 nM, 10 nM, 5nM, 1 nM, 500 pM, 100 pM or less. In some embodiments, the FBS, e.g.,¹⁰Fn3 domain, binds to a desired target with a K_(d) between 1 pM and 1µM, between 100 pM and 500 nM, between 1 nM and 500 nM, or between 1 nMand 100 nM. In exemplary embodiments, the ¹⁰Fn3 moiety bindsspecifically to a target that is not bound by a wild-type ¹⁰Fn3 domain,particularly the wild-type human ¹⁰Fn3 domain having, e.g., SEQ ID NO:1-8.

In certain embodiments, an FBS moiety comprises an amino acid sequencethat is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,or 99% identical to an amino acid sequence selected from the group ofsequences consisting of SEQ ID NOs: 1-16, and the FBS binds specificallyto a target, e.g., with a K_(d) of less than 500 nM, 100 nM, 50 nM, 10nM, 5 nM, 1 nM, 500 pM, 100 pM or less. The FBS moiety may compriseamino acid changes (or alterations) in one or more loops and one or morescaffold regions.

In some embodiments, one or more residues of the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ IDNO: 1) may be substituted so as to disrupt integrin binding. In someembodiments, the FG loop of the polypeptides provided herein does notcontain an RGD integrin binding site. In one embodiment, the RGDsequence is replaced by a polar amino acid-neutral amino acid-acidicamino acid sequence (in the N-terminal to C-terminal direction). Incertain embodiments, the RGD sequence is replaced with SGE or RGE.

In some embodiments, the amino acid sequences of the N-terminal and/orC-terminal regions of an FBS moiety are modified by deletion,substitution or insertion relative to the amino acid sequences of thecorresponding regions of ¹⁰Fn3 domains comprising, e.g., SEQ ID NO: 1.

In certain embodiments, the amino acid sequence of the first 1, 2, 3, 4,5, 6, 7, 8 or 9 residues of SEQ ID NO: 1 may be modified or deleted inthe polypeptides provided herein relative to the sequence of thecorresponding amino acids in the wild-type human ¹⁰Fn3 domain having SEQID NO: 1. In exemplary embodiments, the amino acids corresponding toamino acids 1-7, 8 or 9 of any one of SEQ ID NOs: 1-16 are replaced withan alternative N-terminal region having from 1-20, 1-15, 1-10, 1-8, 1-5,1-4, 1-3, 1-2, or 1 amino acids in length. Exemplary alternativeN-terminal regions include (represented by the single letter amino acidcode) M, MG, G, MGVSDVPRDL (SEQ ID NO: 24) and GVSDVPRDL (SEQ ID NO:25), or N-terminal truncations of any one of SEQ ID NOs: 24 or 25. Othersuitable alternative N-terminal regions include, for example,X_(n)SDVPRDL (SEQ ID NO: 26), X_(n)DVPRDL (SEQ ID NO: 27), X_(n)VPRDL(SEQ ID NO: 28), X_(n)PRDL (SEQ ID NO: 29), X_(n)RDL (SEQ ID NO: 30),XnDL (SEQ ID NO: 31), or X_(n)L, wherein n = 0, 1 or 2 amino acids,wherein when n = 1, X is Met or Gly, and when n = 2, X is Met-Gly. Whena Met-Gly sequence is added to the N-terminus of a ¹⁰Fn3 domain, the Mwill usually be cleaved off, leaving a G at the N-terminus. In otherembodiments, the alternative N-terminal region comprises the amino acidsequence MASTSG (SEQ ID NO: 32).

As further described herein, in some embodiments, the first seven oreight residues (i.e., residues 1-7 or 1-8) of SEQ ID NO: 1 are deleted,generating a ¹⁰Fn3 domain having the amino acid sequence of, e.g., SEQID NO: 8. Additional sequences may also be added to the N- or C-terminusof a ¹⁰Fn3 domain having the amino acid sequence of any one of SEQ IDNOs: 1-16. For example, in some embodiments, the N-terminal extensionconsists of an amino acid sequence selected from the group consistingof: M, MG, and G. For example, any one of SEQ ID NO: 1-16 may bepreceded by M, MG, or G.

In certain embodiments, an FBS moiety is based on an Fn3 repeat otherthan the 10^(th) repeat of the type III domain of fibronectin, e.g.,human fibronectin. For example, an FBS moiety may be similar to any ofthe other fibronectin type III repeats, e.g., the 1^(st), 2^(nd),3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th), 11^(th),12^(th), 13^(th), 14^(th), 15^(th), 16^(th), 17^(th), and 18^(th) Fn3repeats. In yet other embodiments, an FBS moiety may be from a moleculeother than fibronectin. Exemplary FBS moieties may be derived fromtenascin, a protein that is composed of 15 Fn3 domains with similarsequence similarities to one another as found in fibronectin. Theserepeats are described, e.g., in Jacobs et al., Protein Engineering,Design & Selection, 25:107 (2012). Based on the homology of the repeatsin the fibronectin molecule and those in the tenascin molecule,artificial molecules based on these homologies have been created.Proteins comprising a consensus amino acid sequence based on thehomology of the domains in the fibronectin molecule are referred to asFibcon and FibconB (WO 2010/093627 and Jacobs et al. (2012) supra.) andthose based on the homology of the domains in the tenascin molecule arereferred to as Tencon. An exemplary Fibcon amino acid sequence comprisesthe following amino acid sequence:MPAPTDLRFTNETPSSLLISWTPPRVQITGYIIRYGPVGSDGRVKEFTVPPSVSSATITGLKPGTEYTISVIALKDNQESEPLRGRVTTGG (FibconB; SEQ ID NO: 33), wherein loopAB consists of amino acids 13-16 (TPSS; SEQ ID NO: 34), loop BC consistsof amino acids 22-28 (TPPRVQI; SEQ ID NO: 35), loop CD consists of aminoacids 38-43 (VGSDGR; SEQ ID NO: 36), loop DE consists of amino acids51-54 (PSVS; SEQ ID NO: 37), loop EF consists of amino acids 60-64(GLKPG; SEQ ID NO: 38) and loop FG consist of amino acids 75-81(KDNQESEP; SEQ ID NO:39). Another Fibcon amino acid sequence comprisesthe following amino acid sequence:LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNGPGEPKELTVPPSSTSVTITGITPGVEYVVSVYALKDNQESPPLVGTCTT (SEQ ID NO: 40; Jacobs et al., supra).Tenascin derived Fn3 proteins include Tencons (WO 2010/051274, WO2010/051310 and WO 2011/137319, which are specifically incorporated byreference herein). An exemplary Tencon protein has the following aminoacid sequence:

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 41; Jacobs et al., supra,and WO 2011/137319),

wherein loop AB consists of amino acids 13-16 (TEDS; SEQ ID NO: 42, loopBC consists of amino acids 22-28 (TAPDAAF; SEQ ID NO: 43), loop CDconsists of amino acids 38-43 (SEKVGE; SEQ ID NO: 44), loop DE consistsof amino acids 51-54 (GSER; SEQ ID NO: 45), loop EF consists of aminoacids 60-64 (GLKPG; SEQ ID NO: 46) and loop FG consists of amino acids75-81 (KGGHRSN; SEQ ID NO: 47).

A Fibcon, FibconB or Tencon moiety, or target binding variants thereof,whether by themselves or linked to a heterologous moiety may be fused asdescribed herein. Fn3 domains from other proteins, e.g., cell surfacehormone and cytokine receptors, chaperonins, and carbohydrate-bindingdomains, may be conjugated as described herein.

FBS proteins or moieties are described, e.g., in WO 2010/093627, WO2011/130324, WO 2009/083804, WO 2009/133208, WO 02/04523, WO2012/016245, WO 2009/023184, WO 2010/051310, WO 2011/020033, WO2011/051333, WO 2011/051466, WO 2011/092233, WO 2011/100700, WO2011/130324, WO 2011/130328, WO 2011/137319, WO 2010/051274, WO2009/086116, WO 09/058379, WO 2013/067029 WO 2012/016245, WO 2014/120891and WO 2014/043344 (all of which are specifically incorporated byreference herein): any of the FBS proteins or moieties described inthese publications may be used as described herein.

In certain embodiments, a protein comprises at least 2 FBS moieties,e.g., the protein comprises a multivalent FBS moiety. For example, amultivalent FBS may comprise 2, 3 or more FBS moieties, e.g., ¹⁰Fn3domains, that are covalently associated. In exemplary embodiments, theFBS moiety is a bispecific or dimeric protein comprising two ¹⁰Fn3domains.

The FBS moieties, e.g., ¹⁰Fn3 domains, in a multivalent protein may beconnected by a polypeptide linker. Exemplary polypeptide linkers includepolypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2amino acids. Suitable linkers for joining the ¹⁰Fn3 domains are thosewhich allow the separate domains to fold independently of each otherforming a three dimensional structure that permits high affinity bindingto a target molecule. Specific examples of suitable linkers includeglycine-serine based linkers, glycine-proline based linkers,proline-alanine based linkers as well as any other linkers describedherein. In some embodiments, the linker is a glycine-proline basedlinker. These linkers comprise glycine and proline residues and may bebetween 3 and 30, 10 and 30, and 3 and 20 amino acids in length.Examples of such linkers include GPG, GPGPGPG (SEQ ID NO: 48) andGPGPGPGPGPG (SEQ ID NO: 49). In some embodiments, the linker is aproline-alanine based linker. These linkers comprise proline and alanineresidues and may be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18amino acids in length. Examples of such linkers include PAPAPA (SEQ IDNO: 50), PAPAPAPAPAPA (SEQ ID NO: 51) and PAPAPAPAPAPAPAPAPA (SEQ ID NO:52).

In some embodiments, the linker is a glycine-serine based linker. Theselinkers comprise glycine and serine residues and may be between 8 and50, 10 and 30, and 10 and 20 amino acids in length. Examples of suchlinkers include GSGSGSGSGS ((GS) ₅; SEQ ID NO: 53), GSGSGSGSGSGS ((GS)₆; SEQ ID NO: 54), GSGSGSGSGSGSGSGSGSGS ((GS) ₁₀; SEQ ID NO: 55),GGGGSGGGGSGGGGS ((G₄S)₄; SEQ ID NO: 56), GGGGSGGGGSGGGGSGGGGSGGGGS((G₄S) ₅; SEQ ID NO: 57), and GGGGSGGGGSGGGSG (SEQ ID NO: 58). Inexemplary embodiments, the linker does not contain any Asp-Lys (DK)pairs.

PmXn Moieties, e.g., Stabilizing Moieties

In certain embodiments, an FBS, e.g., a ¹⁰Fn3 moiety, is linked at itsC-terminus to a moiety consisting of PmXn, wherein P is a proline, X isany amino acid, m is an integer that is at least 1 and n is 0 or aninteger that is at least 1, and P is N-terminal to X. The PmXn moietymay be linked directly to the C-terminal amino acid of a ¹⁰Fn3 moiety,e.g., to its 94^(th) amino acid (based on amino acid numbering of SEQ IDNO: 1). The PmXn moiety may be linked via a peptide bond to the 94^(th)amino acid of a ¹⁰Fn3 moiety. A PmXn moiety may be linked to a ¹⁰Fn3moiety having an amino acid sequence that is homologous to that of SEQID NO: 1 or comprises, consists essentially of or consists of an aminoacid sequence shown in Table 1 or 2. A single proline residue at the endof SEQ ID NO: 1 is referred to as “95Pro” or “Pro95” or “P95” or “95P”.Exemplary ¹⁰Fn3 moieties linked to a PmXn moiety include the following:

LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTPmXn (SEQ ID  NO: 59)

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTPmXn (SEQ ID NO: 60)

In PmXn, m may be 1, 2, 3 or more. For example, m may be 1-3 or m may be1-2. “n” may be 0, 1, 2, 3 or more, e.g., n may be 1-3 or 1-2.

As further described herein, these ¹⁰Fn3 moieties may be modified tobind to a target (and form FBS moieties), by modifying the amino acidsequence of one or more loop and/or one or more β-strands. FBS moietiesthat are linked to PmXn are referred to herein as “modified FBSmoieties”. Accordingly, provided herein are proteins comprising a FBSmoiety comprising an amino acid sequence that is at least about 50%,60%, 70%, 80%, 90%, or 95% identical to SEQ ID NO: 59 or 60, wherein theprotein comprises PmXn, and wherein the FBS binds specifically to atarget (other than through the RGD domain).

In PmXn, n may be 0, in which case, the C-terminal amino acid of theprotein is Pm, e.g., P. In certain embodiments, n is not 0, and may be,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. For example, n may be from0-10, 0-5, 0-3, 1-10, 1-5, 1-3 or 1-2. However, more than 10 amino acidsmay be linked to the proline. For example, in a tandem FBS moiety or aFBS moiety fused to another polypeptide, the C-terminal amino acid ofthe FBS moiety may be linked to one or more prolines, and the lastproline is linked to the second FBS moiety or to the heterologousmoiety. Therefore, in certain embodiments, n may be an integer rangingfrom 0-100, 0-200, 0-300, 0-400, 0-500 or more.

In certain embodiments, PmXn comprises a cysteine. For example, thefirst amino acid after the proline may be a cysteine, and the cysteinemay be the last amino acid in the molecule or the cysteine may befollowed by one or more amino acids. The presence of a cysteine permitsthe conjugation of heterologous moieties to the FBS moiety, e.g.,chemical moieties, e.g., PEG. Exemplary PmXn moieties comprising acysteine include: PmCXn, wherein C is a cysteine. Another example isPmXn₁CXn₂, wherein n₁ and n₂ are independently 0 or an integer that isat least 1. For example, n₁ may be 1 and n₂ may be 1, 2, 3, 4 or 5.

Exemplary PmXn moieties include those listed in Table 3.

TABLE 3 Exemplary PmXn moieties Moieties with 1 proline Moieties with 2prolines P PP PI PPI PC PPC PID PPID (SEQ ID NO: 114) PIE PPIE (SEQ IDNO: 115) PIDK (SEQ ID NO: 61) PPIDK (SEQ ID NO: 62) PIEK (SEQ ID NO: 63)PPIEK (SEQ ID NO: 64) PIDKP (SEQ ID NO: 65) PPIDKP (SEQ ID NO: 66) PIEKP(SEQ ID NO: 67) PPIEKP (SEQ ID NO: 68) PIDKPS (SEQ ID NO: 69) PPIDKPS(SEQ ID NO: 70) PIEKPS (SEQ ID NO: 71) PPIEKPS (SEQ ID NO: 72) PIDKPC(SEQ ID NO: 73) PPIDKPC (SEQ ID NO: 74) PIEKPC (SEQ ID NO: 75) PPIEKPC(SEQ ID NO: 76) PIDKPSQ (SEQ ID NO: 77) PPIDKPSQ (SEQ ID NO: 78) PIEKPSQ(SEQ ID NO: 79) PPIEKPSQ (SEQ ID NO: 80) PIDKPCQ (SEQ ID NO: 81)PPIDKPCQ (SEQ ID NO: 82) PIEKPCQ (SEQ ID NO: 83) PPIEKPCQ (SEQ ID NO:84) PHHHHHH (SEQ ID NO: 85) PPHHHHHH (SEQ ID NO: 86) PCHHHHHH (SEQ IDNO: 87) PPCHHHHHH (SEQ ID NO: 88)

Any of the PmXn moieties, e.g., those shown in Table 3 may be followedby a histidine tail, e.g., 6xHis tag, or other tag. This does notexclude that a histidine tail may be included in PmXn.

The addition of a PmXn moiety to an FBS moiety enhances one or morecharacteristics of the FBS moiety. For example, as shown in theExamples, it enhances the thermostability of a ¹⁰Fn3 moiety relative tothe moiety that is not linked to a PmXn moiety. The improvement ofthermostability is expected to improve other desirable properties suchas solubility, proper folding and expression level. For example, asshown in the Example, the presence of a PmXn moiety at the C-terminus ofan FBS enhances the solubility of the FBS relative to the FBS that isnot linked to a PmXn moiety.

Thus, in certain embodiments, the Tm of an FBS, e.g., a ¹⁰Fn3, moiety isenhanced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15° C. relative to the FBS moiety that is not linked to a PmXn moiety.For example, the Tm may be increased from 1-30, 1-25, 1-20, 1-15, 1-10,or 1-5° C. relative to the FBS moiety that is not linked to a PmXnmoiety. Tm may be measured by Thermal Scanning Fluorescence (TSF), e.g.,as follows. Protein samples, e.g., HTPP samples, are normalized to 0.2mg/ml in PBS. 1 µl of SYPRO® orange dye diluted 1:40 with PBS is addedto 25 µl of each sample and the plate is sealed with a clear 96-wellmicroplate adhesive seal. Samples are scanned using a BioRad RT-PCRmachine by ramping the temperature from 25° C. - 95° C., at a rate of 2degrees per minute. The data is analyzed using BioRad CFX manager 2.0software. Tm may also be measured by Differential Scanning Calorimetry(DSC) as follows. A 0.5 mg/ml solution is scanned in a VP-CapillaryDifferential Scanning calorimeter (GE Microcal) by ramping thetemperature from 15° C. to 110° C. at a rate of 1 degree per minuteunder 70 p.s.i pressure. The data is analyzed versus a control run ofthe appropriate buffer using a best fit using Origin Software (OriginLabCorp).

In certain embodiments, the solubility of an FBS moiety is enhanced bylinking it to a PmXn moiety. Such a molecule may exist at aconcentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

PKE Moieties

FBS moieties may be linked to PKE moieties to extend the half-life ofthe FBS moieties. Exemplary PKE moieties include human serum albumin;proteins that bind to human serum albumin (e.g., an FBS binding to HSAor ABD); Fc; or any portion or variant thereof; and PEGs. These moietiesmay be linked N-terminal or C-terminal to the PmXn moiety and/or theFBS.

Cysteine Conjugated Labels and Therapeutics

In certain embodiments, an FBS moiety linked to a PmXn moiety (andreferred to a “modified FBS moiety”), wherein at least one or more theamino acids “X” is a cysteine, is linked through the one or morecysteines, to a heterologous moiety, such as a labeling moiety, abiologically active moiety (e.g., a therapeutic agent) or a bindingmoiety.

FBS moieties described herein may be conjugated through a C-terminalcysteine to a therapeutic agent to form an immunoconjugate such as anFBS-drug conjugate (FBS-DC; also “adnectin-drug conjugate”).

In an FBS-DC, the FBS is conjugated to a drug, with the FBS functioningas a targeting agent for directing the FBS-DC to a target cellexpressing its antigen, such as a cancer cell. Preferably, the antigenis a tumor associated antigen, i.e., one that is uniquely expressed orover-expressed by the cancer cell. Once there, the drug is released,either inside the target cell or in its vicinity, to act as atherapeutic agent. For a review on the mechanism of action and use ofdrug conjugates as used with antibodies, e.g., in cancer therapy, seeSchrama et al., Nature Rev. Drug Disc., 5:147 (2006).

Suitable therapeutic agents for use in drug conjugates includeantimetabolites, alkylating agents, DNA minor groove binders, DNAintercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclearexport inhibitors, proteasome inhibitors, topoisomerase I or IIinhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors,antibiotics, and anti-mitotic agents. In an FBS-DC, the FBS andtherapeutic agent preferably are conjugated via a linker cleavable suchas a peptidyl, disulfide, or hydrazone linker. More preferably, thelinker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 89), Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The FBS-DCs can beprepared according to methods similar to those described in U.S. Pat.Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; andWO 08/103693; U.S. Pat. Publication Nos. 2006/0024317; 2006/0004081; and2006/0247295; the disclosures of which are incorporated herein byreference. A linker can itself be linked, e.g., covalently linked, e.g.,using maleimide chemistry, to a cysteine of the PmXn moiety, wherein atleast one X is a cysteine. For example, a linker can be covalentlylinked to an FBS-PmXn, wherein at least one X is a cysteine. Forexample, a linker can be linked to an FBS-PmCn, wherein P is a proline,C is a cysteine, and m and n are integers that are at least 1, e.g.,1-3. Ligation to a cysteine can be performed as known in the art usingmaleimide chemistry (e.g., Imperiali, B. et al., Protein Engineering:Nucleic Acids and Molecular Biology, Vol. 22, pp. 65-96, Gross, H.J.,ed. (2009)). For attaching a linker to a cysteine on an FBS, the linkermay, e.g., comprise a maleinimido moiety, which moiety then reacts withthe cysteine to form a covalent bond. In certain embodiments, the aminoacids surrounding the cysteine are optimized to facilitate the chemicalreaction. For example, a cysteine may be surrounded by negativelycharged amino acid for a faster reaction relative to a cysteine that issurrounded by a stretch of positively charged amino acids (EP 1074563).

For cancer treatment, the drug preferably is a cytotoxic drug thatcauses death of the targeted cancer cell. Cytotoxic drugs that can beused in FBS-DCs include the following types of compounds and theiranalogs and derivatives:

-   (a) enediynes such as calicheamicin (see, e.g., Lee et al., J. Am.    Chem. Soc., 109:3464, 3466 (1987)) and uncialamycin (see, e.g.,    Davies et al., WO 2007/038868 A2 (2007) and Chowdari et al., U.S.    Pat. No. 8,709,431 B2 (2012));-   (b) tubulysins (see, e.g., Domling et al., U.S. Pat. No. 7,778,814    B2 (2010); Cheng et al., U.S. Pat. No. 8,394,922 B2 (2013); and Cong    et al., U.S. Publication No. 2014/0227295 A1;-   (c) CC-1065 and duocarmycin (see, e.g., Boger, U.S. Pat. No.    6,5458,530 B1 (2003); Sufi et al., U.S. Pat. No. 8,461,117 B2    (2013); and Zhang et al., U.S. Publication No. 2012/0301490 A1    (2012));-   (d) epothilones (see, e.g., Vite et al., U.S. Publication No.    2007/0275904 A1 (2007) and U.S. Pat. No. RE42,930 E (2011));-   (e) auristatins (see, e.g., Senter et al., U.S. Pat. No. 6,844,869    B2 (2005) and Doronina et al., U.S. Pat. No. 7,498,298 B2 (2009));-   (f) pyrrolobenzodiazepine (PBD) dimers (see, e.g., Howard et al.,    U.S. Publication Nos. 2013/0059800 A1 (2013) and 2013/0028919 A1    (2013); and WO 2013/041606 A1 (2013)); and-   (g) maytansinoids such as DM1 and DM4 (see, e.g., Chari et al., U.S.    Pat. No. 5,208,020 (1993) and Amphlett et al., U.S. Pat. No.    7,374,762 B2 (2008)).

In certain embodiments, an FBS-PmXn, wherein at least one X is acysteine, is linked to a labeling or detectable moiety for use, e.g., invitro or in vivo detection or imaging.

Detectable labels can be any of the various types used currently in thefield of in vitro diagnostics, including particulate labels includingmetal sols such as colloidal gold, isotopes such as I¹²⁵ or Tc⁹⁹presented for instance with a peptidic chelating agent of the N₂S₂, N₃Sor N₄ type, chromophores including fluorescent markers, biotin,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. A biotinylated antibody would then bedetectable by avidin or streptavidin binding. Suitable enzyme labelsinclude horseradish peroxidase, alkaline phosphatase and the like. Forinstance, the label can be the enzyme alkaline phosphatase, detected bymeasuring the presence or formation of chemiluminescence followingconversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl)phenyl phosphate (CSPD), as well as CDP and CDP-STAR® or otherluminescent substrates well-known to those in the art, for example thechelates of suitable lanthanides such as Terbium(III) and Europium(III).

Detectable moieties that may be used include radioactive agents, suchas: radioactive heavy metals such as iron chelates, radioactive chelatesof gadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ¹⁸F ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ¹²⁴I, ⁸⁶Y, ⁸⁹Zr, ⁶⁶Ga,⁶⁷Ga, ⁶⁸Ga, ⁴⁴Sc, ⁴⁷Sc, ¹¹C, ¹¹¹In, ¹ ¹⁴In, ¹¹⁴In, ¹²⁵I, ¹²⁴I, ¹³¹I,¹²³I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr,¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁵Ac, or¹⁵³Sm.

The detection means is determined by the chosen label. Appearance of thelabel or its reaction products can be achieved using the naked eye, inthe case where the label is particulate and accumulates at appropriatelevels, or using instruments such as a spectrophotometer, a luminometer,a fluorometer, and the like, all in accordance with standard practice.

A detectable moiety may be linked to a cysteine according to methodsknown in the art. When the detectable moiety is a radioactive agent,e.g., those described further herein, the detectable moiety is linked toan FBS through a chelating agent that is reactive with cysteines, suchas a maleimide containing chelating agent, such as maleimide-NODAGA ormaleimide-DBCO. Maleimide-NODAGA or maleimide-DBCO can be reacted with acysteine on the C-terminus of an FBS (e.g., through the PmXn moiety,wherein at least one X is a cysteine), to yield FBS-NODAGA or FBS-DBCO,respectively. Any one of the following chelating agents may be usedprovided that it comprises, or can be modified to comprise, a reactivemoiety that reacts with cysteines: DFO, DOTA and its derivatives(CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A), TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P,MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTA, NETA,TACN-TM, DTPA, 1B4M-DTPA, CHX-A″-DTPA, TRAP (PRP9), NOPO, AAZTA andderivatives (DATA), H2dedpa, H4octapa, H₂azapa, Hsdecapa, H₆phospa,HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA basedchelating agents, and close analogs and derivatives thereof.

In certain embodiments, an FBS is labeled with a PET tracer and used asan in vivo imaging agent. For example, an FBS may be labeled with thePET tracer ⁶⁴Cu. ⁶⁴Cu may be linked to an FBS with a C-terminal cysteinewith a chelating agent, such as maleimide-NODAGA.

Exemplary Molecules

In certain embodiments, a protein comprises (i) an FBS moiety comprisingan amino acid sequence that is at least about 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:1-16; and (ii) PmXn, wherein P is a proline, X is any amino acid, m isan integer of at least 1 and n is 0 or an integer of at least 1, whereinthe protein binds specifically to a target (e.g., with a K_(d) of lessthan 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM or less,as determined, e.g., by Surface Plasmon Resonance (SPR), such asBiacore) and wherein the PmXn moiety improves at least one property ofthe FBS moiety relative to a protein consisting of the unmodified FBSmoiety.

In certain embodiments, an enhanced property conferred by a PmXn moietyis enhanced protein stability, e.g., an increase in melting temperature(Tm) of at least 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C.,9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 1-15° C., 2-15°C., 3-15° C., 5-15° C., 5-15° C., 1-10° C., 2-10° C., 3-10° C., 4-10° C.or 5-10° C. Tm may be determined, e.g., by Thermal Scanning Fluorescence(TSF) or Differential scanning calorimetry (DSC). “Enhanced Tm” refersto a statistically significant enhancement of the Tm.

In certain embodiments, a protein comprising an FBS moiety and a PmXnmoiety is present in a composition, e.g., a pharmaceutical composition,at a concentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml,50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

In certain embodiments, a protein comprising an FBS moiety and a PmXnmoiety is present in a composition, e.g., a pharmaceutical composition,mostly as a monomer, e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% ofthe protein in the composition is in monomeric form. Degree ofmonomericity of a protein solution may be determined by Size ExclusionChromatography (SEC), e.g., by using a Superdex column (GE Healthcare)on an Agilent 1100 or 1200 HPLC system with UV detection at A₂₁₄ nm andA₂₈₀ nm and with fluorescence detection (excitation =280 nm, emission =350 nm). A buffer of 100 mM sodium sulfate, 100 mM sodium phosphate, 150mM sodium chloride, (e.g., pH 6.8) at an appropriate flow rate of theSEC column may be employed. Gel filtration standards (Bio-RadLaboratories, Hercules, CA) are used for molecular weight calibration.

In certain embodiments, a protein comprising an FBS moiety and a PmXnmoiety has a biological activity that is as least as strong as that ofthe unmodified FBS moiety. Biological activity can be binding affinityto a target or a biological activity in an assay, e.g., the ability todestroy tumor cells. In certain embodiments, the biological activity ofthe protein is within 5%, 10%, 25%, 50%, 100%, 2 fold or more of theactivity of the unmodified FBS moiety.

A protein comprising an FBS and a PmXn moiety may also comprise acombination of the above characteristics. For example, a protein may besoluble at concentrations of up to 50 mg/ml, be present at least 90% inmonomeric form, and/or have a biological activity that is at least aspotent as that of the unmodified FBS.

When referring to an enhanced property, the enhancement is astatistically significant enhancement.

Nucleic Acid-Protein Technology

One way to rapidly make and test FBS domains with specific bindingproperties is the nucleic acid-protein technology of Adnexus, aBristol-Myers Squibb Company. Such in vitro expression and taggingtechnology, termed Profusion, that exploits nucleic acid-protein fusions(RNA- and DNA-protein fusions) may be used to identify novelpolypeptides and amino acid motifs that are important for binding toproteins. Nucleic acid-protein technology is a technology thatcovalently couples a protein to its encoding genetic information. For adetailed description of the RNA-protein technology and fibronectin-basedscaffold protein library screening methods see Szostak et al., U.S. Pat.Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; 6,518,018;PCT Publication Nos. WO 00/34784; WO 01/64942; WO 02/032925; and Robertset al., Proc Natl. Acad. Sci., 94:12297-12302 (1997), hereinincorporated by reference.

Vectors and Polynucleotides Embodiments

Nucleic acids encoding any of the various proteins comprising an FBSmoiety a PmXn moiety disclosed herein may be synthesized chemically,enzymatically or recombinantly. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al.,Protein Expr. Purif., 26(1):96-105 (October 2002); Connell, N.D., Curr.Opin. Biotechnol., 12(5):446-449 (October 2001); Makrides et al.,Microbiol. Rev., 60(3):512-538 (September 1996); and Sharp et al.,Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989),or Ausubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. The DNA encoding the polypeptide isoperably linked to suitable transcriptional or translational regulatoryelements derived from mammalian, viral, or insect genes. Such regulatoryelements include a transcriptional promoter, an optional operatorsequence to control transcription, a sequence encoding suitable mRNAribosomal binding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a polypeptide with a heterologous polypeptide,which is preferably a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. The heterologous signal sequence selected preferably is onethat is recognized and processed (i.e., cleaved by a signal peptidase)by the host cell. For prokaryotic host cells that do not recognize andprocess a native signal sequence, the signal sequence is substituted bya prokaryotic signal sequence selected, for example, from the group ofthe alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxinII leaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces alpha-factor leaders), oracid phosphatase leader, the C. albicans glucoamylase leader, or thesignal described in PCT Publication No. WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available. TheDNA for such precursor regions may be ligated in reading frame to DNAencoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 µ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC® No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC® 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein. Promoters suitable for use with prokaryotichosts include the phoA promoter, beta-lactamase and lactose promotersystems, alkaline phosphatase, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding the protein.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT (SEQ ID NO: 109) region where N may be anynucleotide. At the 3′ end of most eukaryotic genes is an AATAAA (SEQ IDNO: 110) sequence that may be the signal for addition of the poly A tailto the 3′ end of the coding sequence. All of these sequences aresuitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP Patent Publication No. 73,657 and PCT Publication Nos. WO2011/124718 and WO 2012/059486. Yeast enhancers also are advantageouslyused with yeast promoters.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the ACTIN® promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature, 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

Transcription of a DNA encoding a protein by higher eukaryotes is oftenincreased by inserting an enhancer sequence into the vector. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature, 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the polypeptide-encoding sequence, but ispreferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the polypeptide. Oneuseful transcription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the proteins. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG® tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, Elsevier, New York(1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct may be introduced into the host cell using amethod appropriate to the host cell, as will be apparent to one of skillin the art. A variety of methods for introducing nucleic acids into hostcells are known in the art, including, but not limited to,electroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified proteins are prepared by culturingsuitable host/vector systems to express the recombinant proteins. TheFBS protein is then purified from culture media or cell extracts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the proteins may be cultured in a varietyof media. Commercially available media such as Ham’s F10 (Sigma),Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), andDulbecco’s Modified Eagle’s Medium ((DMEM), (Sigma)) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enzymol., 58:44 (1979), Barnes et al., Anal. Biochem.,102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; PCT Publication Nos. WO 90/03430; WO 87/00195;or U.S. Pat. No. RE30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as Gentamycin drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-freetranslation systems. For such purposes the nucleic acids encoding theprotein must be modified to allow in vitro transcription to produce mRNAand to allow cell-free translation of the mRNA in the particularcell-free system being utilized (eukaryotic such as a mammalian or yeastcell-free translation system or prokaryotic such as a bacterialcell-free translation system).

Proteins can also be produced by chemical synthesis (e.g., by themethods described in Solid Phase Peptide Synthesis, Second Edition, ThePierce Chemical Co., Rockford, IL (1984)). Modifications to the proteincan also be produced by chemical synthesis.

The proteins disclosed herein can be purified by isolation/purificationmethods for proteins generally known in the field of protein chemistry.Non-limiting examples include extraction, recrystallization, salting out(e.g., with ammonium sulfate or sodium sulfate), centrifugation,dialysis, ultrafiltration, adsorption chromatography, ion exchangechromatography, hydrophobic chromatography, normal phase chromatography,reversed-phase chromatography, gel filtration, gel permeationchromatography, affinity chromatography, electrophoresis, countercurrentdistribution or any combinations of these. After purification, proteinsmay be exchanged into different buffers and/or concentrated by any of avariety of methods known to the art, including, but not limited to,filtration and dialysis.

The purified protein is preferably at least 85% pure, more preferably atleast 95% pure, and most preferably at least 98% or 99% pure. Regardlessof the exact numerical value of the purity, the protein is sufficientlypure for use as a pharmaceutical product.

Exemplary Uses

Modified FBS proteins may be used for any purpose for which FBS proteinsthat are not modified by the addition of a PmXn moiety can be used.

In one aspect, the application provides proteins comprising a modifiedFBS moiety that is useful in the treatment of disorders. The diseases ordisorders that may be treated will be dictated by the bindingspecificity of the FBS moiety. As described herein, modified FBSmoieties may be designed to bind to any target of interest. Exemplarytargets include, for example, TNF-alpha, VEGFR2, PCSK9, IL-23, EGFR andIGF1R. Merely as an example, modified FBS moieties that bind toTNF-alpha may be used to treat autoimmune disorders such as rheumatoidarthritis, inflammatory bowel disease, psoriasis, and asthma. ModifiedFBS proteins described herein may also be used for treating cancer.

In certain embodiments, a method for treating a subject having adisease, e.g., cancer, comprises administering to the subject a modifiedFBS-drug conjugate.

Provided herein are methods for administering proteins to a subject. Insome embodiments, the subject is a human. In some embodiments, theproteins are pharmaceutically acceptable to a mammal, in particular ahuman. A “pharmaceutically acceptable” composition refers to acomposition that is administered to an animal without significantadverse medical consequences. Examples of pharmaceutically acceptablecompositions include compositions comprising FBS moieties that lack theintegrin-binding domain (RGD) and compositions that are essentiallyendotoxin or pyrogen free or have very low endotoxin or pyrogen levels.

Other uses of the modified FBS proteins described herein include theiruse in in vitro or in vivo detection assays. For example, they may beused for detecting a target molecule in a sample. A method may comprisecontacting the sample with a modified FBS described herein, wherein saidcontacting is carried out under conditions that allow FBS-target complexformation; and detecting said complex, thereby detecting said target insaid sample. Detection may be carried out using any art-recognizedtechnique, such as, e.g., radiography, immunological assay, fluorescencedetection, mass spectroscopy, or surface plasmon resonance. The samplemay be from a human or other mammal. For diagnostic purposes,appropriate agents are detectable labels that include radioisotopes, forwhole body imaging, and radioisotopes, enzymes, fluorescent labels andother suitable antibody tags for sample testing.

In certain embodiments, the modified FBS described herein are useful ina variety of diagnostic and imaging applications. In certainembodiments, a modified FBS is labeled with a moiety that is detectablein vivo and such labeled FBS may be used as in vivo imaging agents,e.g., for whole body imaging. For example, in one embodiment, a methodfor detecting a tumor comprising a given antigen in a subject comprisesadministering to the subject a modified FBS linked to a detectablelabel, and following an appropriate time, detecting the label in thesubject.

An FBS imaging agent may be used to diagnose a disorder or diseaseassociated with increased levels of a given antigen, for example, acancer in which a tumor selectively overexpresses the antigen. In asimilar manner, a modified FBS that binds specifically to a givenantigen can be used to monitor antigen levels in a subject being treatedfor a condition associated with the antigen. The modified FBS may beused with or without modification, and may be labeled by covalent ornon-covalent attachment of a detectable moiety.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the proteins described herein, wherein thecomposition is essentially endotoxin and/or pyrogen free.

Therapeutic formulations comprising proteins are prepared for storage bymixing the described proteins having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Osol, A., ed., Remington’s Pharmaceutical Sciences, 16th Edition(1980)), in the form of aqueous solutions, lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as Tween, PLURONIC® or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The proteins may also be entrapped in microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Osol, A., ed., Remington’sPharmaceutical Sciences, 16th Edition (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the proteins described herein, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT® (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated proteins remain in the body for a long time,they may denature or aggregate as a result of exposure to moisture at37° C., resulting in a loss of biological activity and possible changesin immunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

While the skilled artisan will understand that the dosage of eachprotein will be dependent on the identity of the protein, the preferreddosages can range from about 10 mg/square meter to about 2000 mg/squaremeter, more preferably from about 50 mg/square meter to about 1000mg/square meter.

For therapeutic applications, the proteins are administered to asubject, in a pharmaceutically acceptable dosage form. They can beadministered intravenously as a bolus or by continuous in over a periodof time, by intramuscular, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The protein may alsobe administered by intratumoral, peritumoral, intralesional, orperilesional routes, to exert local as well as systemic therapeuticeffects. Suitable pharmaceutically acceptable carriers, diluents, andexcipients are well known and can be determined by those of skill in theart as the clinical situation warrants. Examples of suitable carriers,diluents and/or excipients include: (1) Dulbecco’s phosphate bufferedsaline, pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. Themethods of the present invention can be practiced in vitro, in vivo, orex vivo.

Administration of proteins, and one or more additional therapeuticagents, whether co-administered or administered sequentially, may occuras described above for therapeutic applications. Suitablepharmaceutically acceptable carriers, diluents, and excipients forco-administration will be understood by the skilled artisan to depend onthe identity of the particular therapeutic agent being co-administered.

When present in an aqueous dosage form, rather than being lyophilized,the protein typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted. For the treatment of disease, the appropriate dosage ofproteins will depend on the type of disease to be treated, the severityand course of the disease, whether the proteins are administered forpreventive or therapeutic purposes, the course of previous therapy, thepatient’s clinical history and response to the fusion, and thediscretion of the attending physician. The protein is suitablyadministered to the patient at one time or over a series of treatments.

Exemplary Embodiments

1. An isolated fibronectin based scaffold (FBS) protein thatspecifically binds to a target, wherein the FBS is a non-naturallyoccurring FBS, and wherein the FBS is linked at its C-terminus to amoiety consisting of the amino acid sequence PmXn, wherein P is proline,X is any amino acid, m is an integer that is at least 1 and n is 0 or aninteger that is at least 1, and wherein the PmXn moiety provides anenhanced property to the FBS protein relative to the FBS protein that isnot linked to the PmXn moiety.

2. The isolated FBS protein of embodiment 1, wherein the moiety consistsof P (m is 1 and n is 0).

3. The isolated FBS protein of embodiment 1, wherein the moiety consistsof PP (m is 2 and n is 0).

4. The isolated FBS protein of embodiment 1, wherein the moiety consistsof PmXn, wherein n is 1-150.

5. The isolated FBS protein of embodiment 1, wherein the moiety consistsof PmXn, wherein n is 1-10.

6. The isolated FBS protein of embodiment 1, wherein the moiety consistsof PmXn, wherein n is 1-5.

7. The isolated FBS protein of embodiment 1, wherein the moiety consistsof PI, PC, PID, PIE, PIDK (SEQ ID NO: 61), PIEK (SEQ ID NO: 63), PIDKP(SEQ ID NO: 65), PIEKP (SEQ ID NO: 67), PIDKPS (SEQ ID NO: 69), PIEKPS(SEQ ID NO: 71), PIDKPC (SEQ ID NO: 73), PIEKPC (SEQ ID NO: 75), PIDKPSQ(SEQ ID NO: 77), PIEKPSQ (SEQ ID NO: 79), PIDKPCQ (SEQ ID NO: 81),PIEKPCQ (SEQ ID NO: 83), PHHHHHH (SEQ ID NO: 87) or PCHHHHHH (SEQ ID NO:86).

8. The isolated FBS protein of any one of embodiments 1-7, wherein theFBS protein is an Fn3 protein.

9. The isolated FBS protein of embodiment 8, wherein the Fn3 protein isa ¹⁰Fn3 protein.

10. The isolated FBS protein of embodiment 9, wherein the ¹⁰Fn3 proteinis a human ¹⁰Fn3 protein.

11. The isolated FBS protein of any one of embodiments 1-10, comprisingan amino acid sequence that is at least 50% identical to SEQ ID NO: 1.

12. The isolated FBS protein of any one of embodiments 1-11, comprisingat least one amino acid mutation in at least one loop or one scaffoldregion.

13. The isolated FBS protein of any one of embodiments 1-12, comprisingthe amino acid sequence

VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 9),

wherein (X)_(u), (X)_(v), (X)_(w), (X)_(x), (X)_(y) and (X)_(z)consistof the wild-type amino acid sequence (SEQ ID NO: 1) or comprise at leastone amino acid difference with the corresponding wild-type sequence, andthe sequence optionally comprises from 1-10 scaffold, N-terminal and/orC-terminal mutations.

14. The isolated FBS protein of any one of embodiments 1-13, comprisingan amino acid sequence that is at least 50% identical to SEQ ID NO: 1,and wherein the C-terminal amino acid residue of the FBS protein isfused to a moiety consisting of PmXn, wherein the FBS protein bindsspecifically to a target with a Kd of less than 500 nM, which target isnot bound by a wild-type ¹⁰Fn3 molecule comprising SEQ ID NO: 1.

15. The isolated FBS protein of embodiment 14, comprising an amino acidsequence that is at least 70% identical to SEQ ID NO: 1.

16. The isolated FBS protein of any one of embodiments 1-15, wherein themoiety consisting of PmXn is linked to the C-terminal amino acid residueof the FBS protein through a peptide bond.

17. The isolated FBS protein of any one of embodiments 1-16, whereinPmXn is P or PC.

18. The isolated FBS protein of any one of embodiments 1-17, comprisingan amino acid sequence that is at least 60% identical to SEQ ID NO: 1,and wherein the C-terminal amino acid residue of the FBS protein islinked to a proline through a peptide bond, and wherein the FBS proteinbinds specifically to a target with a Kd of less than 500 nM, whichtarget is not bound by a wild-type ¹⁰Fn3 molecule comprising SEQ ID NO:1.

19. The isolated FBS protein of any of embodiments 1-18, wherein atleast one X of PmXn is a cysteine and wherein the cysteine

20. The isolated FBS protein of embodiment 19, wherein the cysteine isconjugated to a heterologous moiety.

21. The isolated FBS protein of embodiment 20, wherein the heterologousmolecule is a detectable moiety.

22. The isolated FBS protein of embodiment 20 or 21, wherein theheterologous molecule is a drug moiety and the drug moiety and the FBSform an FBS-drug conjugate.

23. The isolated FBS protein of any one of embodiments 1-22, wherein theenhanced property conferred by the PmXn moiety is enhanced stability.

24. The isolated FBS protein of embodiment 23, wherein enhancedstability is an increase in Tm of at least 1° C., 2° C., 3° C., 4° C.,5° C. or more.

The following representative Examples contain important additionalinformation, exemplification and guidance which can be adapted to thepractice of this invention in its various embodiments and theequivalents thereof. These examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit its scope.

EXAMPLES Example 1: Thermostability Enhancement by Adding a Proline atthe C-terminus of ¹⁰Fn3 Molecules

In nature, ¹⁰Fn3 is a part of a long string of fibronectin type IIIdomains; the domain immediately downstream of ¹⁰Fn3 is named ¹¹Fn3. Thesequence below shows the wild-type sequences of the ¹⁰Fn3 (in italics)and ¹¹Fn3 (in bold) domains and the junction between them (underlined).

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS QMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVT (SEQ ID NO: 90)

Based on the crystal and NMR structures of ¹⁰Fn3 and of the sequencealignment between ¹⁰Fn3 and ¹¹Fn3 (Dickinson et al., “Crystal structureof the tenth type III cell adhesion module of human fibronectin”, J.Mol. Biol., 36:1079-1092 (1994)), the first two residues of thissequence, RT, are part of the final beta strand of ¹⁰Fn3 (“strand G”),and the remainder of the sequence, EIDKPSQ (SEQ ID NO: 113), is aflexible linker between structured tenth and eleventh fibronectin typeIII domains.

The C-terminus of engineered protein domains based on ¹⁰Fn3 is oftenmodified from the wild-type sequence to facilitate its cloning,expression, and purification. During a survey of different engineeredC-termini of Adnectins it was found that a mutation from RTE to RTP hasled to increased thermostability of several different Adnectins. Table 4lists different C-termini used in this study.

Table 4: Wild-type linker between ¹⁰Fn3 and ¹¹Fn3 and the engineeredAdnectin C-terminal sequences that were compared in this study. The“short engineered C-terminus with P and His-tag” and the “shortengineered C-terminus with PC” contain the RTE to RTP mutation.

Descriptor Sequence Purpose Comments Wild-type junction between ¹⁰Fn3and ¹¹Fn3 RTEIDKPSQ (SEQ ID NO: 91) natural human sequence contains RTELong engineered C-terminus with His-tag RTEIEKPSQH₆ (SEQ ID NO: 92) H₆allows purification by metal-chelate chromatography contains RTE; DK->EKmutation reduced sensitivity to proteases Short engineered C-terminuswith P and His-tag RTPH₆ (SEQ ID NO: 93) H₆ allows purification bymetal-chelate chromatography contains RTP Short engineered C-terminuswith EC RTEC (SEQ ID NO: 94) C allows site-specific modification usingmaleimide chemistry contains RTE; no purification tag Short engineeredC-terminus with PC RTPC (SEQ ID NO: 95) C allows site-specificmodification using maleimide chemistry contains RTP; no purification tag

The effect of the mutation from RTE to RTP on thermostability ofAdnectins was tested by comparing several pairs of Adnectins thatdiffered in their C-termini. Table 5 describes the pairs of Adnectinsused in this study, including their names, targets, and thermodynamicproperties. Adnectin 1 is an Adnectin that binds to target X. All theAdnectins listed were selected from high-complexity libraries usingPROfusion (mRNA-display) technology, and had their C-terminire-engineered by site-directed mutagenesis. Full protein sequences areset forth below.

Table 5: Clone name, C-terminus, target, and melting temperature (asdetermined by differential scanning calorimetry at 0.5 mg/mL, in PBS, pH7.4). Adnectins PRD-1414 and PRD-1417 were conjugated to 40 kDa,2-branched PEG (NOF, Cat. #GL2-400MA01)

Protein ID (RTE) C-terminus Protein ID (RTP) C-terminus Target T_(m)(RTE) T_(m) (RTP) Increase in T_(m) of RTP- over RTE-containing proteinADX_2382_D09 RTEIEKPSQH₆ (SEQ ID NO: 92) ADX_5484_A03 RTPH₆ (SEQ ID NO:93) PCSK9 84° C. 87° C. +3° C. ADX_2987_H07 RTEIEKPSQH₆ (SEQ ID NO: 92)ADX_5484_A04 RTPH₆ (SEQ ID NO: 93) Myostatin 56° C. 57° C. +1° C.PRD-1414 RTEC-PEG (SEQ ID NO: 94) PRD-1417 RTPC-PEG (SEQ ID NO: 95)PCSK9 64° C. 76° C. +12° C. Adnectin 1 RTEIEKPSQH₆ (SEQ ID NO: 94)Adnectin 1 RTPH₆ (SEQ ID NO: 95) X 61° C. 64° C., 72° C. +3° C., +11° C.

Amino acid sequences of proteins used in this Example:

ADX_2382_D09:

MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTEIDKPSQHHHHHH* (SEQ ID NO: 96)

ADX_5484_A03:

MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTPHHHHHH* (SEQ ID NO: 97)

ADX_2987_H07:

MGVSDVPRDLEVVAATPTSLLISWTLPHAGRAHYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTTTKVIHYKPISINYRTEIDKPSQHHHHHH* (SEQ ID NO: 97)

ADX_5484_A04:

MGVSDVPRDLEVVAATPTSLLISWTLPHAGRAHYYRITYGETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTTTKVIHYKPISINYRTPHHHHHH* (SEQ ID NO: 98)

PRD-1414:

MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTEC* (SEQ ID NO: 99)

PRD-1417:

MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTPC* (SEQ ID NO: 100)

The “*” in each sequence above denotes the stop codon, and theC-terminus of each Adnectin.

The N-terminal M was removed during bacterial expression of eachprotein. The C residues in purified PRD-1414 and PRD-1417 protein wereconjugated to PEG before characterization of each protein.

As shown in Table 5, Adnectins with C-termini that contain a proline inplace of the wild-type glutamic acid at the C-terminus (RTE to RTPmutation) show a higher thermostability. This increase in stability wasobserved for several examples when a longer C-terminus modeled on thenatural linker between human ¹⁰Fn3 and ¹¹Fn3 was replaced with a shorterengineered C-terminus containing only the proline and a hexahistidinepurification tag. Enhanced thermostability was also observed in a ¹⁰Fn3protein having a short engineered C-terminus and a polyethylene glycol(PEG) upon the addition of a C-terminal proline. The increase instability may be attributed to the presence of a proline in thisspecific position.

Example 2: Stabilization of a Second PCSK9 Adnectin with a C-terminalProline

This Example shows that the thermostability of another PCSK9 adnectinmolecule is also enhanced by the addition of a C-terminal proline.

In this Example, the C-terminus of pegylated (40 kDa branched PEG) PCSK9adnectin ADX_2013_E01 was modified from NYRTEIEKPCQ (SEQ ID NO: 101) toNYRTPC (SEQ ID NO: 102) and thermostability measured by TSF. The aminoacid sequence of this adnectin is provided in WO 2011/130354. Theresults indicate that the pegylated adnectin with the NYRTEIEKPCQ (SEQID NO: 101) C-terminus has a Tm of 70° C., whereas the pegylatedadnectin with the NYRTPC (SEQ ID NO: 102) C-terminus has a Tm of 76° C.

Thus, the thermostability of this PCSK9 adnectin is enhanced by 6° C. bythe presence of a C-terminal proline.

Example 3: Comparison of Various C-termini on Thermostability

This Example shows a comparison of the thermostability of adnectins withor without a proline at their C-terminus as well as adnectins having 2prolines at their C-terminus.

Two of the adnectins described in Example 1 (ADX_2392_D09 binding toPCSK9 and ADX_2987_H07 binding to myostatin) and one additional Adnectinbinding to a different target (Adnectin 1, binding to target X) weremodified at their C-terminus, as indicated in Table 6, and theirthermostability and % monomer were determined. An Adnectin (“Adnectin1”) to a different target (X) was also modified with the same C-terminalsequences. Thermostability was determined by TSF and % Monomer wasdetermined by SEC.

TABLE 6 Percent monomer and thermostability of adnectins with variousC-termini Parent \ C-term NYRTEIEKPSQH₆ (SEQ ID NO: 103) NYRTH₆ (SEQ IDNO: 104) NYRTPH₆ (SEQ ID NO: 105) NYRTPPH₆ (SEQ ID NO: 106) ADX_2392_D09PCSK9 >99% 84° C. >99% 83° C. >99% 87° C. 99% 87° C. ADX_2987_H07Myostatin 99% 56° C. >99% 51° C. >99% 57° C. >99% 57° C. Adnectin 1Target X ~ 8% 61° C. >99% 58° C. >98% 64° C.,72° C. >98% 64° C.,72° C.

The results, which are set forth in Table 6, show that the identity ofthe C-terminus has no significant effect on the % monomer, however, ithas an effect on the melting temperature (Tm). As described in Example1, NYRTPH₆ (SEQ ID NO: 105) increases the thermostability relative toNYRTH₆ (SEQ ID NO: 104) for both adnectins: by 4° C. for the PCSK9adnectin and by 6° C. for the myostatin adnectin. In addition, thepresence of a second proline provides a stabilizing effect that issimilar to that provided by a single proline.

Thus, the presence of one or two C-terminal prolines enhances thethermostability of adnectins relative to the same molecule without theC-terminal proline(s).

Example 4: Addition of a C-Terminal Proline Enhances Solubility

This Example demonstrates that the presence of a C-terminal prolineenhances the solubility of a tandem adnectin relative to the same tandemadnectin without the C-terminal proline.

A tandem adnectin, i.e., two adnectins each binding to a differenttarget linked by a linker, and containing either an NYRTE (SEQ ID NO:107) C-terminus or an NYRTP (SEQ ID NO: 108) C-terminus were expressedin E. coli BLR cells and fermented at 30° C. The titer of the tandemwithout the proline was 1.2 g/L soluble protein and 2.8 g/L of totalprotein, indicating that the expressed protein was 43% soluble. Thetiter of the tandem with the proline was 2.56 g/L soluble and 2.61 g/Ltotal. Even after renaturing the insoluble fraction of the tandemwithout the proline, and obtaining a solution that was 98% pure andmonomeric at 2.5 mg/mL, this protein composition did not showsignificant binding to its target, suggesting that it may not beproperly refolded.

Thus, considering that the only difference between the two proteins wasthe presence of an E versus a P at the C-terminus, the results suggeststhat the presence of a proline provides enhanced solubility of thetandem.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books,GENBANK® Accession numbers, SWISS-PROT® Accession numbers, or otherdisclosures) in the Background, Detailed Description, Brief Descriptionof the Drawings, and Examples is hereby incorporated herein by referencein their entirety.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1-24. (canceled)
 25. An isolated multivalent fibronectin based scaffold(FBS) protein comprising two or more ¹⁰Fn3 domains, wherein each of the¹⁰Fn3 domains comprises an amino acid sequence that is at least 70%identical to SEQ ID NO: 1, and wherein (i) the C-terminal amino acid atthe position corresponding to position 94 of SEQ ID NO: 1 of at leastone ¹⁰Fn3 domain is covalently linked through a peptide bond to a moietyconsisting of the amino acid sequence PmXn, wherein P is proline, X isany amino acid, m is an integer that is at least 1 and n is 0 or aninteger that is at least 1; and (ii) the PmXn moiety provides anenhanced property to the multivalent FBS protein relative to the FBSprotein that is not linked to the PmXn moiety.
 26. The isolatedmultivalent FBS protein of claim 25, wherein the moiety covalentlylinked to the at least one ¹⁰Fn3 domain consists of P (m is 1 and n is0).
 27. The isolated multivalent FBS protein of claim 25, wherein themoiety covalently linked to the at least one ¹⁰Fn3 domain consists of PP(m is 2 and n is 0).
 28. The isolated multivalent FBS protein of claim25, wherein the moiety covalently linked to the at least one ¹⁰Fn3domain consists of PmXn, wherein n is 1-10.
 29. The isolated multivalentFBS protein of claim 25, wherein the moiety covalently linked to the atleast one ¹⁰Fn3 domain consists of PmXn, wherein n is 1-5.
 30. Theisolated multivalent FBS protein of claim 25, wherein the PmXn moietycovalently linked to the at least one ¹⁰Fn3 domain consists of PI, PC,PID, PIE, PIDK (SEQ ID NO: 61), PIEK (SEQ ID NO: 63), PIDKP (SEQ ID NO:65), PIEKP (SEQ ID NO: 67), PIDKPS (SEQ ID NO: 69), PIEKPS (SEQ ID NO:71), PIDKPC (SEQ ID NO: 73), PIEKPC (SEQ ID NO: 75), PIDKPSQ (SEQ ID NO:77), PIEKPSQ (SEQ ID NO: 79), PIDKPCQ (SEQ ID NO: 81), PIEKPCQ (SEQ IDNO: 83), PHHHHHH (SEQ ID NO: 87) or PCHHHHHH (SEQ ID NO: 86).
 31. Theisolated FBS protein of claim 25, wherein each of the least ¹⁰Fn3domains comprises the amino acid sequenceLEVVAA(X)uLLISW(X)vYRITY(X)wFTV(X)xATISGL(X)yYTITVY A(X)ZISINYRT (SEQ IDNO: 16), wherein (X)u, (X)v, (X)w, (X)x, (X)y and (X)z consist of thewild-type amino acid sequence (SEQ ID NO: 1) or comprise at least oneamino acid difference with the corresponding wild-type sequence.
 32. Theisolated multivalent FBS protein of claim 25, wherein PmXn is P or PC.33. The isolated multivalent FBS protein of claim 25, wherein at leastone X of PmXn is a cysteine.
 34. The isolated multivalent FBS protein ofclaim 33, wherein the cysteine is conjugated to a heterologous moiety.35. The isolated multivalent FBS protein of claim 34, wherein theheterologous molecule is a detectable moiety.
 36. The isolatedmultivalent FBS protein of claim 34, wherein the heterologous moleculeis a drug moiety and the drug moiety and the multivalent FBS proteinform an FBS-drug conjugate.
 37. The isolated FBS protein of claim 25,wherein the enhanced property conferred by the PmXn moiety is enhancedstability.
 38. The isolated FBS protein of claim 37, wherein enhancedstability is an increase in Tm of at least 1° C., 2° C., 3° C., 4° C.,5° C. or more.
 39. The isolated multivalent FBS protein of claim 33,wherein the PmXn moiety is PmCXn or PmXn₁CXn₂, wherein C is cysteine andn₁ and n₂ are independently 0 or an integer that is at least
 1. 40. Theisolated multivalent FBS protein of claim 32, wherein PmXn is P.
 41. Theisolated multivalent FBS protein of claim 32, wherein PmXn is PC.
 42. Anucleic acid encoding the multivalent FBS protein of claim
 25. 43. Avector comprising the nucleic acid of claim
 42. 44. A host cellcomprising the nucleic acid of claim 42.